Bilirubin is a degradation product of hemoglobin. In a healthy individual, bilirubin released from aged or damaged red blood cells in the body is excreted or degraded into other derivatives. In some cases, however, an abnormal amount of bilirubin occurs within the body in the case of excessive hemolysis or liver failure. There is evidence that excessive amounts of bilirubin in the blood can lead to an undesirable increase in bilirubin concentration within the body cells which interferes with various cellular processes. The clinical significance of bilirubin determination, then, in tests for liver and other related organ functions, is apparent.
In human body fluids such as bile and serum, bilirubin exists in several different forms, these forms commonly being referred to in the art as conjugated bilirubin (B.sub.c, both mono- and diconjugated forms), unconjugated bilirubin (B.sub.u, also known as indirect bilirubin), and delta bilirubin (also known as biliprotein). The total bilirubin content (B.sub.T), represents the sum of all forms of bilirubin.
A variety of colorimetric assays for bilirubin are known. For example, U.S. Pat. No. 4,069,017 describes an assay for bilirubin carried out on a dry multilayer analytical element containing an interactive mordant in a reagent layer which binds to bilirubin thereby producing a detectable product. The mordant also enhances the molar absorptivity of bilirubin and causes a spectral shift in the unconjugated moiety making possible the simultaneous analysis of both conjugated and unconjugated bilirubin by reading reflectance density at 400 and 460 nm. The element also comprises a porous spreading layer and a radiation-blocking layer. Chromophores which can cause spectral interference, such as hemoglobin and delta bilirubin, are retained in the spreading layer above the radiation blocking layer. The bilirubin species, Bu and Bc, migrate through the radiation blocking layer to bind with the mordant. The interactive mordant is dispersed in a binder material such as gelatin or its derivatives.
Unfortunately, gelatin and its derivatives have a slight color change over time in the 400 to 460 nm region of the spectrum, making for poor stability of the system.
A significant advance in the art is described in U.S. Pat. No. 4,788,153 (issued Nov. 29, 1988 to Detwiler). The assay for bilirubin described therein is carried out on an analytical element substantially free of gelatin in the reagent layer. An alternative polymer, poly(acrylamide-co-N-vinylpyrrolidone), was used as the reagent layer vehicle.
However, this polymer is not crosslinkable and the structural integrity of the element cannot be maintained during the analysis, resulting in interferences due to hemoglobin and deltabilirubin.
It is therefore desirable to obtain for use in the reagent layer binder materials that do not absorb light in the 400 to 460 nm range of the spectrum, and which maintain structural integrity.